A simple and colorimetric method utilizing cell-free toehold switch sensors for the detection of Chlamydia trachomatis, Ureaplasma urealyticum and Neisseria gonorrhoeae.

Background: Sexually transmitted infections (STIs) rank among the most prevalent acute infectious conditions and remain a major global public health concern. Notable STI pathogens include Chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), and Neisseria gonorrhoeae (NG). Early detection and diagnosis are crucial for controlling the spread of STIs.

Results: In this study, we utilized toehold switches integrated with a cell-free system to develop a simple, colorimetric, sensitive, specific and rapid method for the parallel detection of CT, UU, and NG. Target DNA and sensor DNA were transcribed into target trigger RNA and toehold switch sensor RNA respectively, within a cell-free transcription system. The binding of target RNA to the toehold switch RNA activated the switch, subsequently initiating the translation of the downstream lacZ gene. The expressed LacZ protein hydrolyzed the substrate chlorophenol red-β-d-galactopyranoside (CPRG), resulting in a color change from yellow to purple, which provided a visible colorimetric output. The three screened sensors exhibited excellent orthogonality without any observed cross-reactivity. By enhancing sensitivity with recombinase polymerase amplification (RPA), we reliably detected NG in clinical samples using this method, with no interference from other pathogens. Moreover, we selected high-performance toehold switch sensor for paper-based detection, further enhancing portability.

Significance: In summary, this technique enables the simple snd sensitive parallel detection of CT, UU, and NG, generating visible colorimetric results without the need for specialized personnel or sophisticated equipment. Given these advantages, this method holds significant potential as a simple and portable diagnostic tool in resource-limited settings or point-of-care testing (POCT) scenarios.